Compositions and methods for inhibiting cd279 interactions

ABSTRACT

Methods and compositions for inhibiting and/or interfering with interactions between (1) programmed Death-1 protein (also known as CD279) and (2) programmed death-ligand 1 (PD-L1) and/or programmed death-ligand 2 (PD-L2) are disclosed. In addition, methods and compositions for increasing IL-2 levels in a cell, and methods and compositions for preventing, treating, or ameliorating the effects of cancer in a subject, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and incorporates by reference, U.S.provisional patent application Ser. No. 62/265,296, filed Dec. 9, 2015.

FIELD OF THE INVENTION

The field of the present invention relates to certain compositions andmethods that are useful in preventing, treating, and/or ameliorating theeffects of various types of cancers. More particularly, the field of thepresent invention relates to certain compositions and methods that maybe used to inhibit PD-1/PD-L1 and/or PD-1/PD-L2 interactions, which mayfurther be useful in preventing, treating, and/or ameliorating theeffects of various types of cancers.

BACKGROUND OF THE INVENTION

The Programmed Death-1 (PD-1) protein, also known as CD279, is aninhibitory receptor that belongs to the CD28 family of receptors. PD-1is expressed on activated T cells, B cells, and myeloid cells andcontains a membrane proximal immune-receptor tyrosine inhibitory motif(ITIM) and a membrane distal tyrosine-based switch motif (ITSM). PD-1has been reported as an immune checkpoint, and to serve an importantrole in down-regulating the immune system by preventing the activationof T cells. Ligation of PD-1 by its ligands has been found to generatean inhibitory signal that results in reduced cytokine production, andreduced T cell survival. Two ligands for PD-1 have previously beenidentified, which have been referred to, in the prior art, as programmeddeath-ligand 1 (PD-L1) (B7-H1) and programmed death-ligand 2 (PD-L2)(B7-DC), and have been shown to down-regulate T cell activation uponbinding to PD-1. Moreover, the interaction between PD-1 and PD-L1 hasalso been found to result in a decrease in tumor infiltratinglymphocytes, a decrease in T-cell receptor mediated proliferation, andimmune evasion by the cancerous cells.

In view of the foregoing, it would be desirable to identify and developone or more peptides that are effective to compete with PD-L1 and/orPD-L2 for binding to PD-1, which may thereby inhibit PD-1/PD-L1 and/orPD-1/PD-L2 interactions. Such inhibitory effects will preferably beuseful in preventing, treating, and/or ameliorating the effects ofvarious types of cancers. As the following will demonstrate, the presentinvention provides such peptides, methods for using such peptides, andother advantages described herein.

SUMMARY OF THE INVENTION

According to certain aspects of the present invention, isolated andpurified peptides are provided that are represented by the amino acidsequences of SEQ ID NO:1, SEQ ID NO:2, derivatives thereof (such as SEQID NO:3-SEQ ID NO:36), and combinations thereof. In addition, theinvention encompasses isolated and purified peptides that aresubstantially homologous to SEQ ID NO:1, SEQ ID NO:2, derivativesthereof, and combinations thereof, such as at least 80%, 90%, or 95%homologous to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3-SEQ ID NO:36, andcombinations thereof. Still further, the present invention encompassesfragments and elongated forms of SEQ ID NO:1, SEQ ID NO:2, andderivatives thereof, as well as nucleic acid sequences and vectors(e.g., viral vectors, such as HSV-1) that encode such peptides orderivatives thereof. Moreover, the present invention encompassespharmaceutical-grade compositions that comprise a peptide consisting ofan amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, derivatives thereof,and combinations thereof (along with fragments thereof, elongated formsthereof, and peptides that are substantially homologous to SEQ ID NO:1,SEQ ID NO:2, derivatives thereof, and combinations thereof).

According to additional aspects of the present invention, methods ofusing the compositions described herein are provided. More particularly,for example, the invention encompasses methods for inhibitinginteractions between PD-1 and PD-L1 in one or more cells byadministering a composition described herein, such as an amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, derivatives thereof (such as thoseof SEQ ID NO:3-SEQ ID NO:36), peptides that are substantially homologousto the foregoing peptides, or pharmaceutical-grade compositions thatcomprise any of such peptides. Likewise, the present inventionencompasses methods for increasing IL-2 levels in one or more cells byadministering a composition described herein, such as an amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, derivatives thereof, combinationsthereof, peptides that are substantially homologous to any of suchpeptides, or pharmaceutical-grade compositions that comprise any of suchpeptides. Still further, the present invention encompasses methods forpreventing, treating, or ameliorating the effects of cancer in a subjectby administering an effective amount of a composition described herein,such as an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, derivativesthereof, combinations thereof, peptides that are substantiallyhomologous to any of such peptides, or pharmaceutical-grade compositionsthat comprise any of such peptides.

According to yet further aspects of the invention, certain derivativesof SEQ ID NO:1 are provided (such as those of SEQ ID NO:3-SEQ ID NO:36),along with peptides that are substantially homologous to the foregoingpeptides. More particularly, such embodiments of the invention includepeptides consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3-SEQ ID NO:36, along with sequences that aresubstantially homologous thereto and pharmaceutical-grade compositionsthat comprise such peptides. Still further, according to relatedembodiments, the invention encompasses (i) methods for inhibitinginteraction between PD-1 and PD-L1 in one or more cells, (ii) methodsfor increasing IL-2 levels in one or more cells, and (iii) methods forpreventing, treating, or ameliorating the effects of cancer in asubject, by administering an effective amount of one or more of suchderivative peptides to the subject.

The above-mentioned and additional features of the present invention arefurther illustrated in the Detailed Description contained herein.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ NO. ID. SequenceDescription  1 WYRMSPSNQT Peptide referred to herein as ″WT″  2TAHPSPSPRSAGQF Peptide referred to herein as ″TF″  3 WYRMSPSNQDA derivative of the WT peptide  4 WYRMSPSNQEA derivative of the WT peptide  5 WYRMSPSNDTA derivative of the WT peptide  6 WYRMSPSNETA derivative of the WT peptide  7 WYRMSPSEQTA derivative of the WT peptide  8 WYRMSPDNQTA derivative of the WT peptide  9 WYRMSPENQTA derivative of the WT peptide 10 WYRMSPPNQTA derivative of the WT peptide 11 WYRMSDSNQTA derivative of the WT peptide 12 WYRMSESNQTA derivative of the WT peptide 13 WYRMAPSNQTA derivative of the WT peptide 14 WYRMQPSNQTA derivative of the WT peptide 15 WYRMMPSNQTA derivative of the WT peptide 16 WYRMPPSNQTA derivative of the WT peptide 17 WYRDSPSNQTA derivative of the WT peptide 18 WYRESPSNQTA derivative of the WT peptide 19 WYIMSPSNQTA derivative of the WT peptide 20 WYLMSPSNQTA derivative of the WT peptide 21 WYYMSPSNQTA derivative of the WT peptide 22 WYVMSPSNQTA derivative of the WT peptide 23 DYRMSPSNQTA derivative of the WT peptide 24 QYRMSPSNQTA derivative of the WT peptide 25 EYRMSPSNQTA derivative of the WT peptide 26 MYRMSPSNQTA derivative of the WT peptide 27 TYRMSPSNQTA derivative of the WT peptide 28 YYRMSPSNQTA derivative of the WT peptide 29 WYRMSWSNQTA derivative of the WT peptide 30 WYRMYPSNQTA derivative of the WT peptide 31 WYRNSPSNQTA derivative of the WT peptide 32 WYTMSPSNQTA derivative of the WT peptide 33 FQGASRPSPSPHATPeptide referred to herein as ″D-FT″ 34 TQNSPSMRYWPeptide referred to herein as ″D-TW″ 35 WNRLSPSNQT Mouse WT peptide 36TRYPSPSPKPEGRF Mouse TF peptide

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing ELISA competition results of WT (SEQ ID NO:1)and TF (SEQ ID NO:2) peptides, and the inhibitory efficacy on PD-1/PD-L1interaction.

FIG. 2A is a graph showing in vitro assay results of WT peptide oninhibiting PD-1/PD-L1 interaction.

FIG. 2B is a graph showing in vitro assay results of TF peptide oninhibiting PD-1/PD-L1 interaction.

FIG. 2C is a graph showing in vitro assay results of a combination of WTand TF peptides on inhibiting PD-1/PD-L1 interaction.

FIG. 3A is a graph showing cell-based assay results of WT peptidetreatment inhibiting PD-1/PD-L1 interaction.

FIG. 3B is a graph showing cell-based assay results of TF peptidetreatment inhibiting PD-1/PD-L1 interaction.

FIG. 3C is a graph showing cell-based assay results of WT and TF peptidetreatment inhibiting PD-1/PD-L1 interaction.

FIG. 4 shows the peptide array results described herein.

FIG. 5A summarizes the results of an ELISA assay, which shows blockingPD-L1 binding to PD-1 by combined PD-L1 blocking peptides.

FIG. 5B summarizes the results of a cell-based assay, which showsdifferent combinations of PD-L1 blocking peptide treatment inhibitingPD-1/PD-L1 interactions.

FIG. 6 summarizes the results of a cytotoxicity assay, which showsblocking PD-L1 via combined peptides enhances cytotoxicity againsttumour cells.

FIG. 7 summarizes the results of an assay that shows the effects ofdifferent PD-L1 blockers on levels of phosphorylated SHP-2.

FIG. 8 summarizes the results of an assay that shows the effects ofadditional PD-L1 blockers on levels of phosphorylated SHP-2.

FIG. 9A summarizes the results of an assay that shows blocking mousePD-L1 binding to mouse PD-1 by mouse peptides.

FIG. 9B shows the difference in amino acid composition between the humanand mouse WT and TF peptides referenced in the Examples.

FIG. 10A summarizes the results of an ELISA assay that shows blockingPD-L1 binding to PD-1 by different forms of PD-L1 blocking peptides.

FIG. 10B summarizes the results of a cell-based assay that showsdifferent forms of PD-L1 blocking peptide treatment inhibitingPD-1/PD-L1 interaction.

FIG. 11 shows the results of an in vivo experiment, showing the effectsof D-TW on mice harbouring tumour cell line LL/2.

FIG. 12 shows the results of an in vivo experiment, showing the effectsof D-TW on tumour size in the mouse model referenced in FIG. 11.

DETAILED DESCRIPTION OF THE INVENTION

The following will describe in detail several preferred embodiments ofthe present invention. These embodiments are provided by way ofexplanation only, and thus, should not unduly restrict the scope of theinvention. In fact, those of ordinary skill in the art will appreciateupon reading the present specification and viewing the present drawingsthat the invention teaches many variations and modifications, and thatnumerous variations of the invention may be employed, used and madewithout departing from the scope and spirit of the invention.

According to certain preferred embodiments of the present invention,isolated and purified peptides are provided that are represented by theamino acid sequences of SEQ ID NO:1, SEQ ID NO:2, combinations thereof,and derivatives thereof (such as those of SEQ ID NO:3-SEQ ID NO:36). Inaddition, the invention encompasses isolated and purified peptides thatare substantially homologous to SEQ ID NO:1, SEQ ID NO:2, combinationsthereof, derivatives thereof, elongated forms thereof, and fragmentsthereof, including peptides that are at least 80%, 90%, or 95%homologous to SEQ ID NO:1, SEQ ID NO:2, derivatives thereof (such asthose of SEQ ID NO:3-SEQ ID NO:36), and combinations thereof. Stillfurther, the present invention encompasses pharmaceutical-gradecompositions that comprise a peptide consisting of an amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, combinations thereof, elongatedforms thereof, fragments thereof, and derivatives thereof (such as thoseof SEQ ID NO:3-SEQ ID NO:36), as well as peptides that are substantiallyhomologous to the foregoing peptides.

The peptides of the present invention may be produced via chemicalsynthesis methods that are well-known in the art. For example, thepeptides may be chemically synthesized using automated Merrifieldtechniques of solid phase synthesis with the α-N H₂ protected by eithert-Boc or F-moc chemistry, using side chain protected amino acids on, forexample, an Applied Biosystems Peptide Synthesizer. The peptides of thepresent invention may also be produced using recombinant DNA technology,including nucleic acid molecules, vectors, and/or host cells. As such,nucleic acid molecules encoding the peptides described herein are alsoencompassed by the present invention. Similarly, vectors, includingexpression vectors, comprising nucleic acid molecules, as well as hostcells containing the vectors, are also encompassed by the presentinvention.

Still further, the invention provides that certain fragments andderivatives of the peptides represented by SEQ ID NO:1-SEQ ID NO:36 areencompassed by the present invention. For example, the peptidesrepresented by SEQ ID NO:1-SEQ ID NO:36 may be modified by substitutingone or more amino acid residues within such peptides, particularlyso-called conservative amino acid substitutions, e.g., an amino acid maybe substituted with an alternative amino acid having similar properties(i.e., substitutions within the same class of amino acid residuessummarized below). For purposes of illustration, substitutions may begrouped into six classes based on common side chain (or “R-group”)properties and the highest frequency of substitution in homologousproteins in nature, as determined, for example, by a standard Dayhofffrequency exchange matrix. The table below represents an example of suchclasses:

Class Residues Description Class I Cysteine Class II Serine, Threonine,Proline, Small aliphatic and OH- Hydroxyproline, Alanine, and group sidechains Glycine Class III Asparagine, Aspartic acid, Neutral andnegatively Glutamic acid, and Glutamine charged side chains capable offorming hydrogen bonds Class IV Histidine, Arginine, and Lysine Basicpolar side chains Class V Isoleucine, Valine, Leucine, and Branchedaliphatic side Methionine chains (except Met) Class VI Phenylalanine,Tyrosine, and Aromatic side chains Tryptophan

In addition, each of the above classes may further include related aminoacid analogs, such as ornithine, homoarginine, N-methyl lysine, dimethyllysine, or trimethyl-lysine in class IV, and a halogenated tyrosine inclass VI.

Furthermore, the peptides described herein may be modified throughsystematic substitution of one or more amino acids with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine), which may beimplemented to generate more stable peptides. Thus, a peptide derivativeor peptidomimetic of the present invention may be all L, all D or mixedD, L peptide, in either forward or reverse order. The presence of anN-terminal or C-terminal D-amino acid increases the in vivo stability ofa peptide, since peptidases cannot utilize a D-amino acid as asubstrate. Reverse-D peptides are peptides containing D-amino acids,arranged in a reverse sequence relative to a peptide containing L-aminoacids. For example, SEQ ID NO:33 represents the reverse, D-amino acidversion of TF (SEQ ID NO:2); and SEQ ID NO:34 represents the reverse,D-amino acid version of WT (SEQ ID NO:1). Thus, the C-terminal residueof an L-amino acid peptide becomes N-terminal for the D-amino acidpeptide, and so forth. Reverse D-peptides retain the same secondaryconformation and therefore similar activity, as the L-amino acidpeptides, but are more resistant to enzymatic degradation in vitro andin vivo, and thus can have greater therapeutic efficacy than theoriginal peptide. Similarly, a reverse L peptide may be generated usingstandard methods where the C-terminus of the parent peptide takes theplace of the N-terminus of the reverse L peptide. The invention providesthat reverse L peptides of L-amino acid peptides that do not havesignificant secondary structure (e.g., short peptides) retain the samespacing and conformation of the side chains of the L-amino acid peptideand, therefore, often have similar activity as the original L-amino acidpeptide. Moreover, a reverse peptide may contain a combination of L-andD-amino acids. The spacing between amino acids and the conformation ofthe side chains may be retained resulting in similar activity as theoriginal L-amino acid peptide.

Still further, the peptides of the present invention may be modified toinclude labels and/or linkers appended thereto, which may be used forimaging purposes, for drug delivery purposes, or to provide additionaltherapeutic payload. For example, the peptides of the present inventionmay be fused to a human IgG C-domain, with the IgG antibody beingdesigned to target and combat tumor cells. Still further, the peptidesof the present invention may be fused or linked to a fragmentcrystallisable (Fc) region of an antibody. In addition, the peptides ofthe present invention include fragments, and elongated forms, of thepeptides represented by SEQ ID NO:1-SEQ ID NO:36. According to yetfurther embodiments, the peptides of the present invention may bechemically and/or structurally modified, e.g., to increase the amount oftime that the peptides will remain active in a biological system (or tootherwise improve the pharmacokinetics of the peptides). For example,the peptides may be modified to include various chemical groups,molecules, or structural adjustments, such as glycosylation,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentto polyethylene glycol (e.g., PEGylation), covalent attachment offlavin, covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, demethylation, formation of covalentcross-links, formylation, gamma carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, ubiquitination, modifications with fattyacids, and various other modifications that may be designed to improvethe pharmacokinetics of the peptides.

The invention provides that the term “peptide,” as used herein, mayinclude not only molecules in which amino acid residues are joined bythe conventional peptide (—CO—NH—) linkages, but also molecules in whichthe peptide bond is reversed. Such retro-inverso peptidomimetics may beproduced using methods known in the art, such as those described inMeziere et al (1997) J. Immunol. 159, 3230-3237 (such approach involvesproducing pseudopeptides containing changes involving the backbone, andnot the orientation of amino acid side chains).

The invention provides that the individual peptides encompassed by theinvention may be administered to a targeted cell (or a plurality ofcells) directly or, alternatively, may be administered indirectly byexpression from an encoding sequence. For example, a polynucleotide(nucleic acid sequence) may be provided to a cell or subject thatencodes a peptide of the invention, such as any of the peptidesrepresented by SEQ ID NO:1-SEQ ID NO:36, as well as modified formsthereof. A peptide of the invention may thus be produced from ordelivered in the form of a polynucleotide which encodes, and is capableof expressing, a peptide of the present invention. Any reference hereinto the use, delivery or administration of a peptide of the invention isintended to include the indirect use, delivery or administration of sucha peptide via expression from a polynucleotide that encodes the peptide.

More particularly, a nucleic acid molecule of the invention may beprovided in isolated or purified form, which encodes a selected peptideof the present invention, which may be transcribed (in the case of DNA)and translated (in the case of mRNA) into a peptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe peptide encoding sequence are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. For the purposes of the present invention, such nucleic acidsequences can include, but are not limited to, cDNA from viral,prokaryotic or eukaryotic mRNA, genomic sequences from viral orprokaryotic DNA or RNA, and even synthetic DNA sequences. The nucleicacid molecules of the present invention may be synthesized according tomethods well-known in the art, as described by way of example inSambrook et al (19104, Molecular Cloning—a laboratory manual; ColdSpring Harbor Press). The nucleic acid molecules of the presentinvention may then be provided in the form of an expression cassette,which includes control sequences operably linked to the peptide-encodingsequence, thus allowing for expression of the applicable peptide of theinvention in vivo in a targeted cell or subject. These expressioncassettes, in turn, are typically provided within vectors, e.g.,plasmids or recombinant viral vectors, such as herpes simplex virus 1(HSV-1). For example, an expression cassette may be administereddirectly to a host cell or subject—or, alternatively, a vectorcomprising a peptide-encoding nucleic acid of the invention may beadministered to a host cell or subject. Methods for delivering exogenousnucleic acid sequences to a host cell or subject are well-known in theart, as described in, for example, U.S. Pat. Nos. 5,399,346, 5,580,859and 5,510,466, which are hereby incorporated by reference.

Still further, the invention provides that isolated and purified formsof the peptides described herein, including various combinations andmixtures of such peptides, may be directly administered to a host cellor subject. For example, such peptides may be administered along withpharmaceutically acceptable (pharmaceutical-grade) carriers, diluentsand adjuvants, such as Dulbecco's phosphate buffered saline, pH about7.4; 0.9% saline (0.9% w/v NaCl); and 5% (w/v) dextrose. Thepharmaceutically acceptable (pharmaceutical-grade) carriers, diluentsand adjuvants used to deliver a peptide of the present invention to atarget cell will, preferably, not induce an immune response in theindividual (subject) receiving the peptide (and will preferably beadministered without undue toxicity). Additional pharmaceuticallyacceptable excipients include, but are not limited to, water, saline,polyethyleneglycol, hyaluronic acid and ethanol. Pharmaceuticallyacceptable salts can also be included therein, e.g., mineral acid salts(such as hydrochlorides, hydrobromides, phosphates, sulfates, and thelike) and the salts of organic acids (such as acetates, propionates,malonates, benzoates, and the like). A thorough discussion ofpharmaceutically acceptable carriers, diluents and adjuvants isavailable in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.1991).

As described above, the present invention encompasses certain methods ofusing the peptides described herein. More particularly, for example, theinvention encompasses methods for inhibiting interactions between PD-1and PD-L1 in one or more cells by administering one or more peptidesdescribed herein to one or more targeted cells, such as an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2, combinations thereof, fragmentsthereof, elongated forms thereof, derivatives thereof (such as those ofSEQ ID NO:3-SEQ ID NO:36), peptides that are substantially homologous tothe foregoing peptides, or pharmaceutical-grade compositions (such asthose described above) that comprise any of such peptides. Likewise, thepresent invention encompasses methods for increasing IL-2 levels in oneor more cells by administering one or more peptides described herein,such as an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,combinations thereof, fragments thereof, elongated forms thereof,derivatives thereof (such as those of SEQ ID NO:3-SEQ ID NO:36),peptides that are substantially homologous to the foregoing peptides, orpharmaceutical-grade compositions that comprise any of such peptides.Still further, the present invention encompasses methods for preventing,treating, or ameliorating the effects of cancer in a subject byadministering an effective amount of one or more peptides describedherein, such as an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,combinations thereof, fragments thereof, elongated forms thereof,derivatives thereof (such as those of SEQ ID NO:3-SEQ ID NO:36),peptides that are substantially homologous to the foregoing peptides, orpharmaceutical-grade compositions that comprise any of such peptides.

As described above, according to yet further embodiments of theinvention, certain derivatives of SEQ ID NO:1 may be used in the methodsreferenced above. More particularly, peptides consisting of (or, inother embodiments, comprising) an amino acid sequence selected from thegroup consisting of SEQ ID NO:3-SEQ ID NO:36, or combinations thereof,fragments thereof, elongated forms thereof, peptides that comprise aminoacid sequences that are substantially homologous thereto, andpharmaceutical-grade compositions that comprise such peptides may beused in the methods referenced above. More particularly, such derivativepeptides may be used to carry out (i) methods for inhibiting interactionbetween PD-1 and PD-L1 in one or more cells, (ii) methods for increasingIL-2 levels in one or more cells, and (iii) methods for preventing,treating, or ameliorating the effects of cancer in a subject, byadministering an effective amount of one or more of such derivativepeptides.

The invention provides that the peptides of the present invention, orthe nucleic acid sequences encoding one or more peptides of the presentinvention, should be administered to a cell or subject in an amount thateffective to achieve the desired endpoint. For particularly, peptides ofthe present invention, or the nucleic acid sequences encoding one ormore peptides of the present invention, should be administered to a cellor subject in an amount that is effective to (i) inhibit interactionbetween PD-1 and PD-L1 in the targeted cells, (ii) increase IL-2 levelsin the targeted cells, and/or (iii) prevent, treat, or ameliorate theeffects of cancer in the targeted subject. The invention provides thatthe peptides described herein may be administered to a cell or subjectin combination or sequence with other agents that exhibit similarproperties (e.g., when the peptides described herein are beingadministered to prevent, treat, or ameliorate the effects of cancer, thepeptides may be administered to a cell or subject in combination or inseries with other agents that are effective to prevent, treat, orameliorate the effects of cancer).

The amount administered to a cell or subject will depend on a variety offactors. For example, in the case of a therapeutic use of the peptides,the amount may vary depending on the size/weight of the subject and theroute of administration, such as orally (e.g. in the form of tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules), parenterally, epicutaneously, subcutaneously, by inhalation,intravenously, intramuscularly, intrasternally, transdermally,intradermally, sublingually, instranasally, buccally or by infusiontechniques. By way of further illustration (and not limitation), theamount of the peptide(s) administered may be in the range of about 5 μgto about 100 mg of peptide; or between about 300 μg and about 1 mg ofpeptide; or amounts in the realm of 300 μg, 350 μg, 400 μg, 450 pg, 500μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950μg or 1 mg of the applicable peptide (or mixture of peptides).

EXAMPLES

Using a peptide array method, two peptides (SEQ ID NO:1 and SEQ ID NO:2)were discovered and chemically synthesized, which are referred to hereinas WT and TF, respectively. SEQ ID NO:1/WT represents the sequence ofWYRMSPSNQT. SEQ ID NO:2/TF represents the sequence of TAHPSPSPRPAGQF. Asthe following will demonstrate, WT and TF (and various derivativesthereof) bind to PD-L1 and prevent PD-L1 from interacting with PD-1.More particularly, as described further below, a series of experimentswere performed to assess the inhibitory efficacy of the WT and TFpeptides (and certain derivatives thereof) on PD-1/PD-L1 interaction (aswell as the impact of such inhibition on IL-2 levels).

FIG. 1 shows ELISA competition results of the WT and TF peptides. Thebinding of the WT or TF peptide to human PD-L1 was tested viacompetition ELISA. Varying concentrations of each peptide (0.008 μg/mlto 10 μg/ml) were mixed with a fixed concentration of recombinant humanPD-1 Fc (10 ng/ml) and bound to a human PD-L1 Fc coated 96-well ImmunoMaxisorp flat bottom plate. Binding was detected via a biotinylatedanti-PD-1 antibody, streptavidin-horseradish peroxidase (HRP), and3,3′,5,5′-Tetramethylbenzidine (TMB) substrate. Absorbance measurementswere collected at 450 nm via a plate reader. The absorbance readingswere plotted against the test peptide concentration. As shown in FIG. 1,as test peptide (WT, TF) concentrations increased, the percent (%)inhibition increased (i.e., the ability of the test peptide to interferewith PD-1/PD-L1 interaction increased).

FIG. 2 shows in vitro assay results of WT, TF, or combined WT and TFpeptides on inhibiting PD-1/PD-L1 interaction (and the resulting effectson IL-2 concentration). 5×10⁴ Jurkat T cells were activated with 1 μg/mlof phytohemagglutinin (PHA) and 50 ng/ml of phorbol 12-myristate13-acetate (PMA) and co-cultured with recombinant human PD-L1 Fc (8μg/ml) plus 1-10 μg/ml of WT (FIG. 2A), 1-10 μg/ml of TF (FIG. 2B), or1-10 μg/ml of combined WT and TF (FIG. 2C) peptide at 37° C. for 48hours. Cell culture supernatant was harvested and interleukin-2 (IL-2)production from Jurkat T cells was assessed by IL-2 ELISA. The numbersshown above each bar in FIG. 2 represent IL-2 concentrations. As shownin FIG. 2, the presence of TF peptide, and the combined WT and TFpeptides, yielded an increase in IL-2 concentration, when administeredalong with PD-L1 Fc.

FIG. 3 shows cell-based assay results of WT, TF, or combined WT and TFpeptide treatments inhibiting PD-1/PD-L1 interaction. In this Example,5×10⁴ Jurkat T cells were activated with 1 μ/ml of PHA and 50 ng/ml ofPMA and co-cultured with 3×10⁵ PD-L1-expressed U87 tumor cells mixedwith different concentrations of WT (FIG. 3A), TF (FIG. 3B), or combinedWT and TF (FIG. 3C) peptide at 37° C. for 48 hours. Cell culturesupernatants were harvested and IL-2 production from the Jurkat T cellswas assessed by IL-2 ELISA. The numbers shown above each bar in FIGS.3A-3C represent IL-2 concentrations. As shown in FIGS. 3A-3C, thepresence of WT peptide, TF peptide, and the combined WT and TF peptidesyielded an increase in IL-2 concentration, when administered along withPD-L1 Fc. Moreover, as shown in FIGS. 3A-3C, as the concentrations ofthe test peptides were increased, an increase in IL-2 concentration wasobserved.

The foregoing assay results demonstrate that both WT and TF peptides areable to block PD-1/PD-L1 interaction. In addition, when the WT and TFpeptides are combined, an enhanced inhibitory effect is observed(relative to a single test peptide treatment). To improve the bindingaffinity of the WT peptide, a single amino acid was substituted at eachposition of the WT peptide and the binding affinities of such derivativepeptides for PD-L1 was examined via peptide arrays. More particularly,peptide array results were generated by incubating derivative testpeptide-containing membrane with 15 μg/mI of negative control human IgGor recombinant human PD-L1 Fc at 4° C., overnight. The resulting signalswere detected by HRP-conjugated anti-human IgG and HRP substrate.Referring now to FIG. 4, each dot shown therein represents a detectedsignal from interactions between a test derivative peptide and humanPD-L1 Fc, with the depth of black color being indicative of the level ofbinding affinity. Based on the peptide array results in FIG. 4, thefollowing sequences of derivative peptides were developed asalternatives to the WT peptide:

SEQ ID. NO. Sequence  3 WYRMSPSNQD  4 WYRMSPSNQE  5 WYRMSPSNDT  6WYRMSPSNET  7 WYRMSPSEQT  8 WYRMSPDNQT  9 WYRMSPENQT 10 WYRMSPPNQT 11WYRMSDSNQT 12 WYRMSESNQT 13 WYRMAPSNQT 14 WYRMQPSNQT 15 WYRMMPSNQT 16WYRMPPSNQT 17 WYRDSPSNQT 18 WYRESPSNQT 19 WYIMSPSNQT 20 WYLMSPSNQT 21WYYMSPSNQT 22 WYVMSPSNQT 23 DYRMSPSNQT 24 QYRMSPSNQT 25 EYRMSPSNQT 26MYRMSPSNQT 27 TYRMSPSNQT 28 YYRMSPSNQT 29 WYRMSWSNQT 30 WYRMYPSNQT 31WYRNSPSNQT 32 WYTMSPSNQT 34 TQNSPSMRYW 35 WNRLSPSNQT

FIG. 5A shows blocking PD-L1 binding to PD-1 by certain PD-L1 blockingpeptides (and certain combinations thereof). More particularly, in thisExample and referring to FIGS. 5A and 5B, WT represents SEQ ID NO:1; TFrepresents SEQ ID NO:2; ET represents SEQ ID NO:25; YT represents SEQ IDNO:28; and TW represents the reverse sequence of WT with D-amino acids,SEQ ID NO:34 (also referred to herein as “D-TW”). Different combinationof such PD-L1 blocking peptides (3 and 10 μM) were mixed withrecombinant human PD-1 Fc and bound to a human PD-L1 Fc coated 96-wellImmuno Maxisorp flat bottom plate. Binding was detected via abiotinylated anti-PD-1 antibody, streptavidin-horseradish peroxidase(HRP), and 3,3′, 5,5′-Tetramethylbenzidine (TMB) substrate. Absorbancemeasurements were collected at 450 nm via a plate reader. The increasedpercent (%) of human PD-1/PD-L1 inhibition was compared to a sample thatwas not provided with a test peptide. The results are summarized in FIG.5A.

FIG. 5B shows cell-based assay results of different PD-L1 blockingpeptide treatment inhibiting PD-1/PD-L1 interaction. In this Example,5×10⁴ Jurkat T cells were activated with 1 μ/ml of PHA and 50 ng/ml ofPMA and co-cultured with 1×10⁵ PD-L1-expressed tumour cells mixed withthe different combinations of PD-L1 blocking peptides at 37° C. for 48hours. Cell culture supernatants were then harvested and IL-2 production(from the Jurkat T cells) was assessed by IL-2 ELISA. As shown in FIGS.5A and 5B, the test PD-L1 blocking peptides (and the tested combinationsthereof) did exhibit inhibition of PD-1/PD-L1 interaction.

FIG. 6 shows blocking PD-L1 through the combined peptides referencedtherein enhances cytotoxicity against tumour cells. In this Example,peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3antibody (OKT-3) plus IL-2 for 24 hours and were subsequently incubatedwith PD-L1 blockers/peptides (the blockers/peptides referenced in FIG.6) and Calcein-AM labelled H460 cells (large cell lung carcinoma) for 24hours. Supernatants were harvested after incubation and then releasedcalcein-AM fluorescence was measured via a microplate reader (emission485, excitation 525). The percentage of cytotoxicity (shown in FIG. 6)was calculated based on the following formula: [(sample reading−minimumrelease)/(maximum release−minimum release)]×100. As shown in FIG. 6, thetest PD-L1 blocking peptides exhibited enhanced cytotoxicity against thesubject tumour cells (relative to the control, “no peptide”), and suchactivity was particularly prominent when such peptides were administeredat 10 μg/ml (versus 3 μg/ml).

Referring now to FIGS. 7 and 8, in this Example, Jurkat cells werestarved in serum-free medium overnight. The cells were subsequentlycultured with different combinations of the PD-L1 blockers/peptides(referenced in FIGS. 7 and 8) or medium alone in a 96-well platepre-coated with an anti-CD3 monoclonal antibody and PD-L1 Fc at 37° C.for 10 minutes. Cold PBS was added into each well to stop the reactionand cell lysates were prepared for blotting with anti-phosphorylatedSHP-2 and anti-Beta-actin antibodies. The expression level ofphosphorylated SHP-2 (a cytoplasmic SH2 domain containing proteintyrosine phosphatase) was standardized based on Beta-actin expression,and the relative expression level of phosphorylated SHP-2 for thedifferent PD-L1 blockers was normalized using medium alone sample. Asshown in FIGS. 7 and 8, a majority of the tested PD-L1 blockers/peptideswere effective to increase the relative expression level ofphosphorylated SHP-2.

FIG. 9A shows the effects of blocking mouse PD-L1 binding to mouse PD1.More specifically, mouse and human WT and TF peptides (3 and 10 μM) weremixed with recombinant mouse PD-1 Fc and bound to a mouse PD-L1 Fccoated 96-well Immuno Maxisorp flat bottom plate. Binding was detectedvia a biotinylated anti-mouse PD-1 antibody, streptavidin-horseradishperoxidase (HRP), and 3,3′, 5,5′ Tetramethylbenzidine (TMB) substrate.Absorbance measurements were collected at 450 nm via a plate reader. Theincreased percent (%) of mouse (and human) PD-1/PD L1 inhibition wascompared to a control (no peptide) sample. The results show specificityof the peptides—specifically, that human peptides do not have the sameblocking effects as mouse peptides for mouse PD-L1. The differencesbetween the human and mouse peptides are illustrated in FIG. 9B.

FIG. 10A shows blocking PD-L1 binding to PD-1 by different forms ofPD-L1 blocking peptides. More particularly, in this Example andreferring to FIGS. 10A and 10B, 3TF3ET represents three copies of TF(SEQ ID NO:2) linked to a linker followed by three copies of ET (SEQ IDNO:25); 3ET3TF represents three copies of ET peptide linked to a linkerfollowed by three copies of TF peptide; and TF-Fc represents TF linkedto a fragment crystallisable (Fc) region of human IgG4. 293FT cells wereinfected with PD-L1 blocking peptide-encoded viral vector for 48 hours.Each peptide (or peptide combination) was expressed and secreted fromthe infected cells (the peptide or peptide combination was secreted viaa fused 5′ signal peptide). Harvested PD-L1 blocking peptide-containedsupernatants from infected 293FT cells or synthetic TF peptide weremixed with recombinant human PD-1 Fc and bound to a human PD-L1 Fccoated 96-well Immuno Maxisorp flat bottom plate. Binding was detectedvia a biotinylated anti-PD-1 antibody, streptavidin, horseradishperoxidase (HRP), and 3,3′, 5,5′-Tetramethylbenzidine (TMB) substrate.Absorbance measurements were collected at 450 nm via a plate reader. Theincreased percent (%) of human PD-1/PD-L1 inhibition was compared to acontrol (no peptide) sample.

FIG. 10B shows cell-based assay results of different forms of PD-L1blocking peptide treatment inhibiting PD-1/PD-L1 interaction. 5×10⁴Jurkat T cells were activated with 1 μ/ml of PHA and 50 ng/ml of PMA andco-cultured with 1×10⁵ PD-L1-expressing tumour cells mixed with PD-L1blocking peptide-contained supernatants or synthetic TF peptide at 37°C. for 48 hours. After 48 hours, cell culture supernatants wereharvested and IL-2 production from the Jurkat T cells was assessed byIL-2 ELISA.

FIG. 11 shows the results of an in vivo experiment, showing the effectsof D-TW on mice harbouring tumour cell line LL/2. More particularly,mice strain C57Bl/6 (male, 4-weeks, initial body weight ˜20-22 g) wereinoculated with tumour cell line LL/2, at S.C. 10⁵ per mouse. After 14days post-implantation, the mice were treated with D-TW orD-TW-Scrambled for 7 days (at 200 mg/kg), via intraperitoneal (IP)injection. FIG. 12 shows the effects of D-TW on tumour size in the micereferenced in FIG. 11. As shown in FIG. 12, it was found that the D-TWpeptide is generally non-toxic to the C57Bl/6 mouse strain. In addition,the results show that the D-TW peptide (at 200 mg/kg) inoculation waseffective to significantly inhibit tumour growth (relative to thecontrol and relative to the D-TW-Scrambled peptide).

The many aspects and benefits of the invention are apparent from thedetailed description, and thus, it is intended for the following claimsto cover all such aspects and benefits of the invention, which fallwithin the scope and spirit of the invention. In addition, becausenumerous modifications and variations will be obvious and readily occurto those skilled in the art, the claims should not be construed to limitthe invention to the exact construction and operation illustrated anddescribed herein. Accordingly, all suitable modifications andequivalents should be understood to fall within the scope of theinvention as claimed herein.

What is claimed is:
 1. A peptide consisting of an amino acid sequencethat is selected from the group consisting of SEQ ID NO:1-SEQ ID NO:36.2. The peptide of claim 1, wherein the peptide consists of one or moreamino acid sequences that are selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:25, SEQ ID NO:28, and SEQ ID NO:34. 3.The peptide of claim 2, wherein the peptide is disposed within apharmaceutical-grade composition.
 4. The peptide of claim 2, wherein thepeptide is linked to a fragment crystallisable (Fc) region of anantibody.
 5. The peptide of claim 3, wherein the peptide is formulatedto be administered to a mammal via local tissue injection, intravenousinjection or intraperitoneal injection.
 6. The peptide of claim 2,wherein the peptide is encoded by a viral or non-viral vector andsecreted into an extracellular space.
 7. The peptide of claim 2, whereinthe peptide is formulated (a) to be administered to a mammal to generatean immune response within said mammal and (b) to raise antibodiesagainst PD-L1.
 8. A method for inhibiting interaction between PD-1 andPD-L1 in a cell by administering one or more peptides to the cell,wherein each of said peptides consists of an amino acid sequence that isselected from the group consisting of SEQ ID NO:1-SEQ ID NO:36.
 9. Themethod of claim 8, wherein each of said peptides consists of one or moreamino acid sequences that are selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:25, SEQ ID NO:28, and SEQ ID NO:34. 10.The method of claim 9, wherein the peptide is disposed within apharmaceutical-grade composition.
 11. The method of claim 10, whereinthe peptide is formulated to be administered to a mammal via localtissue injection, intravenous injection or intraperitoneal injection.12. The method of claim 9, wherein the peptide is encoded by a viral ornon-viral vector and secreted into an extracellular space.
 13. A methodfor preventing, treating, or ameliorating the effects of cancer in asubject by administering one or more peptides to the subject, whereineach of said peptides consists of one or more amino acid sequences thatare selected from the group consisting of SEQ ID NO:1-SEQ ID NO:36. 14.The method of claim 13, wherein each of said peptides consists of one ormore amino acid sequences that are selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:25, SEQ ID NO:28, and SEQ ID NO:34.15. The method of claim 14, wherein the peptide is disposed within apharmaceutical-grade composition.
 16. The method of claim 15, whereinthe peptide is formulated to be administered to a mammal via localtissue injection, intravenous injection or intraperitoneal injection.17. The method of claim 14, wherein the peptide is encoded by a viral ornon-viral vector and secreted into an extracellular space.